AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-compleme...AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2(scAAV2)vector technologies.METHODS:An scAAV2 vector with C3 transferase(C3)as the reporter gene(scAAV2-C3)was selected.The scAAV2-C3 vectors were driven by Ch3L1(scAAV2-Ch3L1-C3),MGP(scAAV2-MGP-C3),enhanced MGP(scAAV2-eMGP-C3)and cytomegalovirus(scAAV2-CMV-C3),respectively.The cultured primary human TM cells were treated with each vector at different multiplicities of infections.Changes in cell morphology were observed by phase contrast microscopy.Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining,real-time quantitative polymerase chain reaction and Western blot.Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively.In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope.Intraocular pressure(IOP)was monitored using a rebound tonometer.Ocular responses were evaluated by slit-lamp microscopy.Ocular histopathology analysis was examined by hematoxylin and eosin staining.RESULTS:In TM cell culture studies,the vectormediated C3 expression induced morphologic changes,disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rhoassociated protein kinase signaling pathway.At the same dose,these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3,but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3.At lowinjected dose,the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGPC3-injected and scAAV2-eMGP-C3-injected eyes.At highinjected dose,significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes.Similar to scAAV2-CMV-C3,scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium.In anterior segment tissues of scAAV2-eMGP-C3-injected eyes,no obvious morphological changes were found except for the TM.Inflammation was absent.CONCLUSION:In scAAV2-transduced TM cells,the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus,but obviously higher than that of MGP.In the anterior chamber of rat eye,the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter,but not by Ch3L1 promoter.These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.展开更多
AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HI...AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P >0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.展开更多
AIM:To observe the changes in Schlemm’s canal(SC),trabecular meshwork(TM),and anterior chamber relevant parameters after small incision lenticule extraction(SMILE) of myopia patients.METHODS:A total of 58 eyes from 3...AIM:To observe the changes in Schlemm’s canal(SC),trabecular meshwork(TM),and anterior chamber relevant parameters after small incision lenticule extraction(SMILE) of myopia patients.METHODS:A total of 58 eyes from 30 patients who underwent SMILE were divided into a low and moderate myopia group(group A,32 eyes) and a high myopia group(group B,26 eyes).The diameter and area of the SC,the width and thickness of TM obtained by CIRRUS HD-OCT5000,and the related anterior chamber parameters obtained by Pentacam anterior segment analysis system,accommodation amplitude(AMP) were observed pre-and postoperatively.The preoperative intraocular pressure(IOP) and postoperative correction of intraocular pressure(IOPcc) were measured.RESULTS:The diameter and area of the SC in the two groups were significantly increased postoperatively(all P<0.01).The TM width of the patients in the two groups were increased at 1mo after surgery(both P<0.01),but the TM thickness did not change(P>0.05).The corneal curvature,central anterior chamber depth,and anterior chamber volume decreased after SMILE surgery(all P<0.01).There was a weak negative correlation between the SC area change and AMP change in group A(r=-0.362,P<0.01).The postoperative IOP decreased after correction by Shah formula(P<0.05).CONCLUSION:SC and TM in myopia patients change in the early postoperative stage of SMILE and the IOP is decline.展开更多
AIM:To investigate the effects of dexamethasone(DEX) and 1-(5-isoquinolinesulfonyl)-homopiperazine(HA1077) on actin cytoskeleton and β-catenin in cultured human trabecular meshwork(HTM) cells.METHODS: The HTM cells w...AIM:To investigate the effects of dexamethasone(DEX) and 1-(5-isoquinolinesulfonyl)-homopiperazine(HA1077) on actin cytoskeleton and β-catenin in cultured human trabecular meshwork(HTM) cells.METHODS: The HTM cells were separated from human eyeball and cultured in vitro.They were divided into control group,DEX(1×10^(-6)mol/L) group,HA1077(3×10^(-5)mol/L)group,and DEX(1×10^(-6)mol/L) and HA1077(3×10^(-5)mol/L)group.Actin cytoskeleton and β-catenin in HTM cells of the four groups were examined by immunofluorescence and Western blot analyses.RESULTS: In DEX group,there were reorganization of actin cytoskeleton and formation of cross linked actin networks(CLANs),which were partially reversed in DEX and HA1077 group.DEX treatment also induced an increased expression of β-catenin,which was obviously reduced in DEX and HA1077 group.Meanwhile,the cultured HTM cells in HA1077 group had lower expression of β-catenin than that in the control group. CONCLUSION: Our results show that HA1077 can reverse the changes of actin organization and expression of β-catenin induced by DEX in cultured HTM cells,suggesting that HA1077 may play an important role in increasing outflow and reducing intraocular pressure.展开更多
In order to explore whether the conventional use of 5 fluorouracil (5 Fu) had any toxic effects on trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and exposed to 5 Fu at different co...In order to explore whether the conventional use of 5 fluorouracil (5 Fu) had any toxic effects on trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and exposed to 5 Fu at different concentrations. The cellular morphology, ultrastructure, mortality and phagocytosis were studied under light microscopy, transmission electron microscopy and methods of Wright’s stain. It was found that the toxic effects of 5 Fu on the cells were in a dose dependent mode. 1×10 -1 mg/ml of 5 Fu caused a large part of cells rounded up, while 1×10 -3 mg/ml of the drug only a rough appearance of the cell surface. Exposure to 1×10 -2 mg/ml of 5 Fu made mitochrone swollen and rough endoplasmic reticulum enlarged, with the cell mortality being 50.5 %. The latex microspheres engulfed in cytoplasm in cells receiving 1×10 -1 and 1×10 -2 mg/ml of 5 Fu were significantly decreased as compared with those in the control group ( P <0.01). It was concluded that the safe concentration of 5 Fu on bovine trabecular meshwork cells was 1×10 -3 mg/ml and the conventional dosage of 5 Fu in clinical practice would not cause injury to trabecular meshwork cells.展开更多
The effect of transforming growth factor β 2 (TGF β 2) on phagocytosis in bovine trabecular meshwork cells in vitro was investigated. After the cultured bovine trabecular meshwork cells were treated with 0 ng/ml, 0....The effect of transforming growth factor β 2 (TGF β 2) on phagocytosis in bovine trabecular meshwork cells in vitro was investigated. After the cultured bovine trabecular meshwork cells were treated with 0 ng/ml, 0.32 ng/ml, 1 ng/ml, 3.2 ng/ml TGF β 2 for 24 h, latex beads were added into the incubation medium, and the numbers of the latex beads in 20 adjacent cells were counted under a microscope 24 h later, after treatment with Wright’s stain. Our results showed that the average numbers of the latex beads in the trabecular meshwork cells treated with TGF β 2 of different concentrations were 53.1±1.7 beads/cell, 56.4±2.9 beads/cell and 77.9±6.5 beads/cell respectinvely, in comparison with 45.5±3.3 beads/cell of the control group. TGF β 2 significantly increased the number of the latex beads phagocytosed by cultured bovine trabecular meshwork cells in a dose dependent manner. TGF β 2 could promote the phagocytosis of bovine trabecular meshwork cells in vitro . It may be involved in the cellularity decrease of the trabecular meshwork in the patients of primary open angle glaucoma through promoting the phagocytosis of trabecular meshwork cells.展开更多
AIM: To investigate changes in extracellular matrix(ECM) gene expression in human trabecular meshwork(HTM) cells in response to mechanical fluid flow stimulation.METHODS: HTM cells were grown on a glass plate coated w...AIM: To investigate changes in extracellular matrix(ECM) gene expression in human trabecular meshwork(HTM) cells in response to mechanical fluid flow stimulation.METHODS: HTM cells were grown on a glass plate coated with 0.02% type Ⅰ collagen(COL) and exposed to shear stress(0, 0.2, 1.0 dyne/cm;) for 12 h.Changes in genes related to the ECM were evaluated by real-time reverse transcriptase-polymerase chain reaction.Phosphorylation of Smad2 protein was investigated by Western blotting.RESULTS: After mechanical stimulation, COL type 4 alpha 2, COL type 6 alpha 1, and fibronectin-1 mRNA were significantly higher than the static control(P<0.05, <0.05, and <0.01, respectively).The metalloproteinase-2 and plasminogen activator inhibitor-1 mRNA were significantly higher than the static control(P<0.05 and <0.01, respectively), while the differences in the tissue inhibitors of metalloproteinases-2 mRNA were not significant.The phosphorylation of Smad2 levels was significantly higher compared to the static control cells.CONCLUSION: Changes in the expressions of genes associated ECM metabolism result in HTM cells after mechanical stimulation.The mechanical stimulation of the aqueous humor to the trabecular meshwork may promote ECM turnover and contribute to intraocular pressure homeostasis.展开更多
AIM:To investigate potential gene changes in trabecular meshwork(TM)induced by dexamethasone(DEX)in steroidinduced glaucoma(SIG).METHODS:The expression data of 24 cases from a public functional genomics data were sort...AIM:To investigate potential gene changes in trabecular meshwork(TM)induced by dexamethasone(DEX)in steroidinduced glaucoma(SIG).METHODS:The expression data of 24 cases from a public functional genomics data were sorted to identify the mechanisms of action of DEX on the TM.The relationships of the differentially expressed genes(DEGs)were enriched using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.In addition,the hub genes were screened by the Search Tool for the Retrieval of Interacting Genes Database(STRING)and Cytoscape tools.Finally,human TM cells(HTMCs)were treated with DEX to preliminarily explore the function of hub genes.RESULTS:Totally 47 DEGs,including 21 downregulated and 26 upregulated genes were identified.The primary enriched results of the DEGs consisted of inflammatory response,extracellular matrix(ECM),negative regulation of cell proliferation,TNF signalling pathway and the regulation of tr yptophan channels by inflammator y mediators.Subsequently,pro-melanin-enriched hormone(PMCH)and Bradykinin B1 receptor(BDKRB1)were screened as hub genes.It is verified in GSE37474 data set.Western blot and quantitative real-time polymerase chain reaction(q PCR)results showed that protein and RNA expression levels of BDKRB1 were significantly decreased after DEX treatment,while PMCH was not significantly changed.CONCLUSION:BDKRB1 may be a key gene involved in SIG onset,providing a suitable therapeutic target for improving the prognosis of SIG patients.展开更多
As the aqueous humor leaves the eye, it first passes through the trabecular meshwork(TM). Increased flow resistance in this region causes elevation of intraocular pressure(IOP), which leads to the occurrence of glauco...As the aqueous humor leaves the eye, it first passes through the trabecular meshwork(TM). Increased flow resistance in this region causes elevation of intraocular pressure(IOP), which leads to the occurrence of glaucoma. To quantitatively evaluate the effect of high IOP on the configuration and hydraulic permeability of the TM, second harmonic generation(SHG) microscopy was used to image the microstructures of the TM and adjacent tissues in control(normal) and high IOP conditions. Enucleated rabbit eyes were perfused at a pressure of 60 mm Hg to achieve the high IOP. Through the anterior chamber of the eye, in situ images were obtained from different depths beneath the surface of the TM. Porosity and specific surface area of the TM in control and high IOP conditions were then calculated to estimate the effect of the high pressure on the permeability of tissue in different depths. We further photographed the histological sections of the TM and compared the in situ images. The following results were obtained in the control condition, where the region of depth was less than55 μm with crossed branching beams and large pores in the superficial TM. The deeper meshwork is a silk-like tissue with abundant fluorescence separating the small size of pores. The total thickness of pathway tissues composed of TM and juxtacanalicular(JCT) is more than 100 μm. After putting a high pressure on the inner wall of the eye, the TM region progressively collapses and decreases to be less than 40 μm. Fibers of the TM became dense, and the porosity at 34 μm in the high IOP condition is comparable to that at 105 μm in the control condition. As a consequent result, the permeability of the superficial TM decreases rapidly from 120 μm2to 49.6 μm2and that of deeper TM decreases from 1.66 μm2to0.57 μm2. Heterogeneity reflected by descent in permeability reduces from 12.4 μm of the control condition to 3.74 μm of the high IOP condition. The persistently high IOP makes the TM region collapse from its normal state, in which the collagen fibers of the TM are arranged in regular to maintain the physiological permeability of the outflow pathway. In the scope of pathologically high IOP, the microstructure of the TM is sensitive to pressure and hydraulic permeability can be significantly affected by IOP.展开更多
AIM:To compare the trabecular meshwork(TM)and iris apoptosis of treated and untreated primary open angle glaucoma(POAG)patients.METHODS:Eight treatment-naive,newly diagnosed(group 1)and 11 medlcaiy treated(group 2)pat...AIM:To compare the trabecular meshwork(TM)and iris apoptosis of treated and untreated primary open angle glaucoma(POAG)patients.METHODS:Eight treatment-naive,newly diagnosed(group 1)and 11 medlcaiy treated(group 2)patients with POAG were included in the study.Each patient underwent a limbus-based trabeculectomy.The TM and peripheral iris specimens were dissected out and were snap-frozen in liquid nitrogen and stored at-80t until they were assayed.Apoptosis in each group was assesed by TUNEL method.RESULTS:The mean patient age was 60.6±5.8 years(53-68 years)vs 58.9±8.9 years(47-70 years)in group 1and group 2(P=0.859).The mean treatment time in group 2 was 22.2±7.3 months(12-34 months).Apoptotic indexes in TM and iris were significantly higher in POAG patients using medication(group 2)compared to treatment-naive POAG patients(group 1)(P=0.004,0.015;respectively).CONCLUSION:Long term administration of topical antiglaucoma medications causes additional toxic effects on TM.展开更多
The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and prima...The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD44-specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44 might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.展开更多
AIM: To determine the effects of a low dose latrunculin (LAT)-A on dexamethasone (Dex)-induced upregulation of extracellular matrix proteins fibronectin (FN) in cultured human trabecular meshwork (HTM) cells.METHODS: ...AIM: To determine the effects of a low dose latrunculin (LAT)-A on dexamethasone (Dex)-induced upregulation of extracellular matrix proteins fibronectin (FN) in cultured human trabecular meshwork (HTM) cells.METHODS: HTM cells were cultured to confluent and incubated with 0.4μmol/L Dex and/or 0.05μmol/L LAT-A.FN expression in HTM cells was evaluated by Western blot and immunofluorescence microscopy.RESULTS: Dex up-regulated FN production in HTM cells,failed to do so when co-incubated with LAT-A.LAT-A decreased production of FN in cultured HTM cells.CONCLUSION: This study indicated that LAT-A may modulate the expression of fibronectin in trabecular meshwork to achieve treatment for steroids and other types of glaucoma.It has an important prospect as an intraocular pressurelowering drug.展开更多
To confirm the existence of heme oxygenase (HO)-carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HTMCs) in vitro, and to evaluate the inductive role ...To confirm the existence of heme oxygenase (HO)-carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HTMCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.展开更多
Purpose:To determine whether cultured human trabecular meshwork cells express CD44 and to discuss their possible relationship with primary open angle glaucoma.Methods:Human trabecular meshwork cells were cultured in D...Purpose:To determine whether cultured human trabecular meshwork cells express CD44 and to discuss their possible relationship with primary open angle glaucoma.Methods:Human trabecular meshwork cells were cultured in DMEM/F12 media. Total RNAs from the cells were extracted with Trizol reagent. Messenger RNA expression of CD44 in human trabecular meshwork cells was examined by using reverse transcriptase-polymerase chain reaction ( RT-PCR ) analysis. Expression of CD44 was confirmed by Western-blotting and immunofluorescent microscopy. Effect of CD44-specific antisense oligonucleotide on adhesion of trabecular meshwork cells to hyaluronate was determined by MTT assay. Results:A single RT-PCR product whose size was 471bp was obtained. A band about 80kD was stained by Western-blot. Immunofluorescent examination of expression of CD44 on the cell surface was positive and reactions were mainly localized in cell membranes. Adhesion of trabecular meshwork cells to hyaluronate was inhibited by CD44-specific antisense oligonucleotide. Conclusions: Cultured human trabecular meshwork cells express CD44. CD44 may play a role in pathogenesis of primary open angle glaucoma. Eye Science 2004;20:52-56.展开更多
Objective:To determine if aquaporin-1 could be detected in cultures of human trabecularshwork cells. Methods: Using primers specific for aquaporin-1, reverse transcription combined withpolymerase chain reaction (RT-PC...Objective:To determine if aquaporin-1 could be detected in cultures of human trabecularshwork cells. Methods: Using primers specific for aquaporin-1, reverse transcription combined withpolymerase chain reaction (RT-PCR) yielded a product and its size with total RNAprepared from the human trabecular meshwork cells. SDS-PAGE and immunoblottingwere also used in this study to detect the specific water channel.Results: The presence of this product and its size (298 base pairs) are consistent withthat of an aquaporin-1 message in these cells. A band of 28 kD in agreement with themolecular size of aquaporin-1 was showed in a film by immunoblotting.Conclusion: The presence of aquaporin-1 in human trabecular meshwork cells, thepredominant cell-type of the primary outflow region of the human eye, suggests that waterchannels may be involved in the movement of aqueous fluid out of the eye. In addition,the existence of aquaporin-1 on cultures of human trabecular meshwork cells provides anin vitro model to study the endogenous expression of aquaporin-1 and its possible role inthe regulation of aqueous outflow.展开更多
Whether cultured bovine trabecular meshwork cells and trabecular tissue ex vivo express insulin like growth factor I (IGF Ⅰ) messenger RNA (mRNA) and protein was investigated. Total RNA of cultured bovine trabecular ...Whether cultured bovine trabecular meshwork cells and trabecular tissue ex vivo express insulin like growth factor I (IGF Ⅰ) messenger RNA (mRNA) and protein was investigated. Total RNA of cultured bovine trabecular meshwork cells as well as trabecular meshwork tissue freshly excised from bovine eyes was extracted, and reverse transcriptase polymerase chain reaction (RT PCR) was used to detect IGF Ⅰ mRNA. RT PCR product was verified by sequencing. Immunohistochemical stain was used to detect IGF Ⅰ protein. The results showed that a single PCR amplified product was obtained, and the sequence was homologous to the known sequence.. IGF Ⅰ immunostain was positive in the cytoplasm of trabecular meshwork cells. It was concluded that trabecular meshwork cells produce IGF Ⅰ and contribute to the presence of IGF Ⅰ in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by IGF Ⅰ not only through paracrine, but also autocrine action. Whether abnormal down regulations in IGF Ⅰ production may contribute to the pathogenesis of primary open angle glaucoma and the possibility of promoting the autocrine action of IGF Ⅰ by trabecular meshwork cells to treat the diesease is worth further investigation.展开更多
Whether transforming growth factor β 2 (TGF β 2) induces apoptosis of human trabecular meshwork cells was investigated in vitro . Cultured 3 5 passage human trabecular meshwork cells were treated with 0 (control), 0...Whether transforming growth factor β 2 (TGF β 2) induces apoptosis of human trabecular meshwork cells was investigated in vitro . Cultured 3 5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF β 2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44) %, (4.43 ±1.17) % and (9.60±2.05) % respectively with different concentrations [1 ng/ml ( P< 0.05), 3.2 ng/ml ( P< 0.01)] of TGF β 2 with the difference being significant between experimental group and control group[(1.41±0.34) %]. It was concluded that TGF β 2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.展开更多
To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative fo...To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTG-ASDON2 were significantly decreased as compared with that of the controls (P<0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.展开更多
The effects of different doses of nitric oxide (NO) on the proliferation and apoptosis of the cultured bovine trabecular meshwork (TM) cells were studied. L arginine and N G nitro L arginine methyl (L NAME) were incub...The effects of different doses of nitric oxide (NO) on the proliferation and apoptosis of the cultured bovine trabecular meshwork (TM) cells were studied. L arginine and N G nitro L arginine methyl (L NAME) were incubated with TM cells for 48 h. In the control group, no medicine was given. In the experimental groups, concentrations of L arginine and L NAME were 1×10 -7 mol/L, 1×10 -6 mol/L, 1×10 -5 mol/L, 1×10 -4 mol/L, 1×10 -3 mol/L and 1×10 -2 mol/L, respectively. NO 2 - in supernate, the proliferation and apoptosis of TM cells and mRNA expression of bcl 2 and bax were measured by Griess reagent, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), MTT assay and in situ hybridization,respectively. The results showed that L arginine with concentration ≥1×10 -4 mol/L could induce apoptosis of the TM cells and inhibit the proliferation of TM cells through increasing the NO levels, down regulating bcl 2 mRNA expression and up regulating bax mRNA expression; L NAME with concentration ≥1×10 -5 mol/L could induce the proliferation of the TM cells through suppressing the production of NO. It was concluded that NO in high level could induce apoptosis of the TM cells and suppress the proliferation of the TM cells.展开更多
基金Supported by the National Natural Science Foundation of China(No.81900829,No.82070963)the Xiamen Medical and Health Guiding Project Fund Project(No.3502Z20214ZD1214)+1 种基金the Guangdong Basic and Applied Basic Research Foundation(No.2019A1515011234)the Science and Technology Innovation Committee of Shenzhen(No.JCYJ20210324125614039)。
文摘AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2(scAAV2)vector technologies.METHODS:An scAAV2 vector with C3 transferase(C3)as the reporter gene(scAAV2-C3)was selected.The scAAV2-C3 vectors were driven by Ch3L1(scAAV2-Ch3L1-C3),MGP(scAAV2-MGP-C3),enhanced MGP(scAAV2-eMGP-C3)and cytomegalovirus(scAAV2-CMV-C3),respectively.The cultured primary human TM cells were treated with each vector at different multiplicities of infections.Changes in cell morphology were observed by phase contrast microscopy.Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining,real-time quantitative polymerase chain reaction and Western blot.Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively.In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope.Intraocular pressure(IOP)was monitored using a rebound tonometer.Ocular responses were evaluated by slit-lamp microscopy.Ocular histopathology analysis was examined by hematoxylin and eosin staining.RESULTS:In TM cell culture studies,the vectormediated C3 expression induced morphologic changes,disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rhoassociated protein kinase signaling pathway.At the same dose,these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3,but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3.At lowinjected dose,the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGPC3-injected and scAAV2-eMGP-C3-injected eyes.At highinjected dose,significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes.Similar to scAAV2-CMV-C3,scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium.In anterior segment tissues of scAAV2-eMGP-C3-injected eyes,no obvious morphological changes were found except for the TM.Inflammation was absent.CONCLUSION:In scAAV2-transduced TM cells,the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus,but obviously higher than that of MGP.In the anterior chamber of rat eye,the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter,but not by Ch3L1 promoter.These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.
基金Supported by Natural Science Foundation of China(No.30901395)the Doctoral Fund of Ministry of Education of China(No.20090142120012,20110142120021)
文摘AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P >0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.
基金Supported by the National Natural Science Foundation of China (No.81974133)。
文摘AIM:To observe the changes in Schlemm’s canal(SC),trabecular meshwork(TM),and anterior chamber relevant parameters after small incision lenticule extraction(SMILE) of myopia patients.METHODS:A total of 58 eyes from 30 patients who underwent SMILE were divided into a low and moderate myopia group(group A,32 eyes) and a high myopia group(group B,26 eyes).The diameter and area of the SC,the width and thickness of TM obtained by CIRRUS HD-OCT5000,and the related anterior chamber parameters obtained by Pentacam anterior segment analysis system,accommodation amplitude(AMP) were observed pre-and postoperatively.The preoperative intraocular pressure(IOP) and postoperative correction of intraocular pressure(IOPcc) were measured.RESULTS:The diameter and area of the SC in the two groups were significantly increased postoperatively(all P<0.01).The TM width of the patients in the two groups were increased at 1mo after surgery(both P<0.01),but the TM thickness did not change(P>0.05).The corneal curvature,central anterior chamber depth,and anterior chamber volume decreased after SMILE surgery(all P<0.01).There was a weak negative correlation between the SC area change and AMP change in group A(r=-0.362,P<0.01).The postoperative IOP decreased after correction by Shah formula(P<0.05).CONCLUSION:SC and TM in myopia patients change in the early postoperative stage of SMILE and the IOP is decline.
基金Supported by the Natural Science Foundation of China (No.81300768 No.81371048+4 种基金 No.81670853)Science and Technology Department of Sichuan Province of China (No.2015HH0031)Health and Family Planning Commission of Sichuan Province of China (No.100539 No. 090505 No.090514)
文摘AIM:To investigate the effects of dexamethasone(DEX) and 1-(5-isoquinolinesulfonyl)-homopiperazine(HA1077) on actin cytoskeleton and β-catenin in cultured human trabecular meshwork(HTM) cells.METHODS: The HTM cells were separated from human eyeball and cultured in vitro.They were divided into control group,DEX(1×10^(-6)mol/L) group,HA1077(3×10^(-5)mol/L)group,and DEX(1×10^(-6)mol/L) and HA1077(3×10^(-5)mol/L)group.Actin cytoskeleton and β-catenin in HTM cells of the four groups were examined by immunofluorescence and Western blot analyses.RESULTS: In DEX group,there were reorganization of actin cytoskeleton and formation of cross linked actin networks(CLANs),which were partially reversed in DEX and HA1077 group.DEX treatment also induced an increased expression of β-catenin,which was obviously reduced in DEX and HA1077 group.Meanwhile,the cultured HTM cells in HA1077 group had lower expression of β-catenin than that in the control group. CONCLUSION: Our results show that HA1077 can reverse the changes of actin organization and expression of β-catenin induced by DEX in cultured HTM cells,suggesting that HA1077 may play an important role in increasing outflow and reducing intraocular pressure.
文摘In order to explore whether the conventional use of 5 fluorouracil (5 Fu) had any toxic effects on trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and exposed to 5 Fu at different concentrations. The cellular morphology, ultrastructure, mortality and phagocytosis were studied under light microscopy, transmission electron microscopy and methods of Wright’s stain. It was found that the toxic effects of 5 Fu on the cells were in a dose dependent mode. 1×10 -1 mg/ml of 5 Fu caused a large part of cells rounded up, while 1×10 -3 mg/ml of the drug only a rough appearance of the cell surface. Exposure to 1×10 -2 mg/ml of 5 Fu made mitochrone swollen and rough endoplasmic reticulum enlarged, with the cell mortality being 50.5 %. The latex microspheres engulfed in cytoplasm in cells receiving 1×10 -1 and 1×10 -2 mg/ml of 5 Fu were significantly decreased as compared with those in the control group ( P <0.01). It was concluded that the safe concentration of 5 Fu on bovine trabecular meshwork cells was 1×10 -3 mg/ml and the conventional dosage of 5 Fu in clinical practice would not cause injury to trabecular meshwork cells.
基金This project was supported by a grant from the NationalNatural Science Foundation of China (No.38970 75 8)
文摘The effect of transforming growth factor β 2 (TGF β 2) on phagocytosis in bovine trabecular meshwork cells in vitro was investigated. After the cultured bovine trabecular meshwork cells were treated with 0 ng/ml, 0.32 ng/ml, 1 ng/ml, 3.2 ng/ml TGF β 2 for 24 h, latex beads were added into the incubation medium, and the numbers of the latex beads in 20 adjacent cells were counted under a microscope 24 h later, after treatment with Wright’s stain. Our results showed that the average numbers of the latex beads in the trabecular meshwork cells treated with TGF β 2 of different concentrations were 53.1±1.7 beads/cell, 56.4±2.9 beads/cell and 77.9±6.5 beads/cell respectinvely, in comparison with 45.5±3.3 beads/cell of the control group. TGF β 2 significantly increased the number of the latex beads phagocytosed by cultured bovine trabecular meshwork cells in a dose dependent manner. TGF β 2 could promote the phagocytosis of bovine trabecular meshwork cells in vitro . It may be involved in the cellularity decrease of the trabecular meshwork in the patients of primary open angle glaucoma through promoting the phagocytosis of trabecular meshwork cells.
基金Supported by JSPS KAKENHI (No.15K10857No.17H00695No.19K18865)。
文摘AIM: To investigate changes in extracellular matrix(ECM) gene expression in human trabecular meshwork(HTM) cells in response to mechanical fluid flow stimulation.METHODS: HTM cells were grown on a glass plate coated with 0.02% type Ⅰ collagen(COL) and exposed to shear stress(0, 0.2, 1.0 dyne/cm;) for 12 h.Changes in genes related to the ECM were evaluated by real-time reverse transcriptase-polymerase chain reaction.Phosphorylation of Smad2 protein was investigated by Western blotting.RESULTS: After mechanical stimulation, COL type 4 alpha 2, COL type 6 alpha 1, and fibronectin-1 mRNA were significantly higher than the static control(P<0.05, <0.05, and <0.01, respectively).The metalloproteinase-2 and plasminogen activator inhibitor-1 mRNA were significantly higher than the static control(P<0.05 and <0.01, respectively), while the differences in the tissue inhibitors of metalloproteinases-2 mRNA were not significant.The phosphorylation of Smad2 levels was significantly higher compared to the static control cells.CONCLUSION: Changes in the expressions of genes associated ECM metabolism result in HTM cells after mechanical stimulation.The mechanical stimulation of the aqueous humor to the trabecular meshwork may promote ECM turnover and contribute to intraocular pressure homeostasis.
文摘AIM:To investigate potential gene changes in trabecular meshwork(TM)induced by dexamethasone(DEX)in steroidinduced glaucoma(SIG).METHODS:The expression data of 24 cases from a public functional genomics data were sorted to identify the mechanisms of action of DEX on the TM.The relationships of the differentially expressed genes(DEGs)were enriched using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.In addition,the hub genes were screened by the Search Tool for the Retrieval of Interacting Genes Database(STRING)and Cytoscape tools.Finally,human TM cells(HTMCs)were treated with DEX to preliminarily explore the function of hub genes.RESULTS:Totally 47 DEGs,including 21 downregulated and 26 upregulated genes were identified.The primary enriched results of the DEGs consisted of inflammatory response,extracellular matrix(ECM),negative regulation of cell proliferation,TNF signalling pathway and the regulation of tr yptophan channels by inflammator y mediators.Subsequently,pro-melanin-enriched hormone(PMCH)and Bradykinin B1 receptor(BDKRB1)were screened as hub genes.It is verified in GSE37474 data set.Western blot and quantitative real-time polymerase chain reaction(q PCR)results showed that protein and RNA expression levels of BDKRB1 were significantly decreased after DEX treatment,while PMCH was not significantly changed.CONCLUSION:BDKRB1 may be a key gene involved in SIG onset,providing a suitable therapeutic target for improving the prognosis of SIG patients.
基金supported by the National Natural Science Foundation of China(Grant Nos.31070840,10802053,and 81201123)the Natural Science Foundation of Beijing,China(Grant No.3122010)+2 种基金the Natural Science Foundation of Guangdong Province,China(Grant No.S2012040006576)the Shenzhen Science and Technology Innovation Committee,China(Grant No.KQCX20120816155352228)the Funding Project for Academic Human Resources Development in Institutions of Higher Learning under the Jurisdiction of Beijing Municipality,China(Grant No.PHR201110506)
文摘As the aqueous humor leaves the eye, it first passes through the trabecular meshwork(TM). Increased flow resistance in this region causes elevation of intraocular pressure(IOP), which leads to the occurrence of glaucoma. To quantitatively evaluate the effect of high IOP on the configuration and hydraulic permeability of the TM, second harmonic generation(SHG) microscopy was used to image the microstructures of the TM and adjacent tissues in control(normal) and high IOP conditions. Enucleated rabbit eyes were perfused at a pressure of 60 mm Hg to achieve the high IOP. Through the anterior chamber of the eye, in situ images were obtained from different depths beneath the surface of the TM. Porosity and specific surface area of the TM in control and high IOP conditions were then calculated to estimate the effect of the high pressure on the permeability of tissue in different depths. We further photographed the histological sections of the TM and compared the in situ images. The following results were obtained in the control condition, where the region of depth was less than55 μm with crossed branching beams and large pores in the superficial TM. The deeper meshwork is a silk-like tissue with abundant fluorescence separating the small size of pores. The total thickness of pathway tissues composed of TM and juxtacanalicular(JCT) is more than 100 μm. After putting a high pressure on the inner wall of the eye, the TM region progressively collapses and decreases to be less than 40 μm. Fibers of the TM became dense, and the porosity at 34 μm in the high IOP condition is comparable to that at 105 μm in the control condition. As a consequent result, the permeability of the superficial TM decreases rapidly from 120 μm2to 49.6 μm2and that of deeper TM decreases from 1.66 μm2to0.57 μm2. Heterogeneity reflected by descent in permeability reduces from 12.4 μm of the control condition to 3.74 μm of the high IOP condition. The persistently high IOP makes the TM region collapse from its normal state, in which the collagen fibers of the TM are arranged in regular to maintain the physiological permeability of the outflow pathway. In the scope of pathologically high IOP, the microstructure of the TM is sensitive to pressure and hydraulic permeability can be significantly affected by IOP.
文摘AIM:To compare the trabecular meshwork(TM)and iris apoptosis of treated and untreated primary open angle glaucoma(POAG)patients.METHODS:Eight treatment-naive,newly diagnosed(group 1)and 11 medlcaiy treated(group 2)patients with POAG were included in the study.Each patient underwent a limbus-based trabeculectomy.The TM and peripheral iris specimens were dissected out and were snap-frozen in liquid nitrogen and stored at-80t until they were assayed.Apoptosis in each group was assesed by TUNEL method.RESULTS:The mean patient age was 60.6±5.8 years(53-68 years)vs 58.9±8.9 years(47-70 years)in group 1and group 2(P=0.859).The mean treatment time in group 2 was 22.2±7.3 months(12-34 months).Apoptotic indexes in TM and iris were significantly higher in POAG patients using medication(group 2)compared to treatment-naive POAG patients(group 1)(P=0.004,0.015;respectively).CONCLUSION:Long term administration of topical antiglaucoma medications causes additional toxic effects on TM.
文摘The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD44-specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44 might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.
基金National Natural Science Foundation of China (No.30772379)Education Department Project of Hunan Province, China(No.04A049)
文摘AIM: To determine the effects of a low dose latrunculin (LAT)-A on dexamethasone (Dex)-induced upregulation of extracellular matrix proteins fibronectin (FN) in cultured human trabecular meshwork (HTM) cells.METHODS: HTM cells were cultured to confluent and incubated with 0.4μmol/L Dex and/or 0.05μmol/L LAT-A.FN expression in HTM cells was evaluated by Western blot and immunofluorescence microscopy.RESULTS: Dex up-regulated FN production in HTM cells,failed to do so when co-incubated with LAT-A.LAT-A decreased production of FN in cultured HTM cells.CONCLUSION: This study indicated that LAT-A may modulate the expression of fibronectin in trabecular meshwork to achieve treatment for steroids and other types of glaucoma.It has an important prospect as an intraocular pressurelowering drug.
文摘To confirm the existence of heme oxygenase (HO)-carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HTMCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.
文摘Purpose:To determine whether cultured human trabecular meshwork cells express CD44 and to discuss their possible relationship with primary open angle glaucoma.Methods:Human trabecular meshwork cells were cultured in DMEM/F12 media. Total RNAs from the cells were extracted with Trizol reagent. Messenger RNA expression of CD44 in human trabecular meshwork cells was examined by using reverse transcriptase-polymerase chain reaction ( RT-PCR ) analysis. Expression of CD44 was confirmed by Western-blotting and immunofluorescent microscopy. Effect of CD44-specific antisense oligonucleotide on adhesion of trabecular meshwork cells to hyaluronate was determined by MTT assay. Results:A single RT-PCR product whose size was 471bp was obtained. A band about 80kD was stained by Western-blot. Immunofluorescent examination of expression of CD44 on the cell surface was positive and reactions were mainly localized in cell membranes. Adhesion of trabecular meshwork cells to hyaluronate was inhibited by CD44-specific antisense oligonucleotide. Conclusions: Cultured human trabecular meshwork cells express CD44. CD44 may play a role in pathogenesis of primary open angle glaucoma. Eye Science 2004;20:52-56.
基金The study was supported by National Nature Science Foun-dation of China(No.39770789 No.39800163)Doctoral Founda-tion of national teaching association(No.9856)Nature Science Foundation of Guangdong(No.980112)
文摘Objective:To determine if aquaporin-1 could be detected in cultures of human trabecularshwork cells. Methods: Using primers specific for aquaporin-1, reverse transcription combined withpolymerase chain reaction (RT-PCR) yielded a product and its size with total RNAprepared from the human trabecular meshwork cells. SDS-PAGE and immunoblottingwere also used in this study to detect the specific water channel.Results: The presence of this product and its size (298 base pairs) are consistent withthat of an aquaporin-1 message in these cells. A band of 28 kD in agreement with themolecular size of aquaporin-1 was showed in a film by immunoblotting.Conclusion: The presence of aquaporin-1 in human trabecular meshwork cells, thepredominant cell-type of the primary outflow region of the human eye, suggests that waterchannels may be involved in the movement of aqueous fluid out of the eye. In addition,the existence of aquaporin-1 on cultures of human trabecular meshwork cells provides anin vitro model to study the endogenous expression of aquaporin-1 and its possible role inthe regulation of aqueous outflow.
基金This projectwas supported by a grant from National Nat-ural Sciences Founction of China (No.38970 75 8)
文摘Whether cultured bovine trabecular meshwork cells and trabecular tissue ex vivo express insulin like growth factor I (IGF Ⅰ) messenger RNA (mRNA) and protein was investigated. Total RNA of cultured bovine trabecular meshwork cells as well as trabecular meshwork tissue freshly excised from bovine eyes was extracted, and reverse transcriptase polymerase chain reaction (RT PCR) was used to detect IGF Ⅰ mRNA. RT PCR product was verified by sequencing. Immunohistochemical stain was used to detect IGF Ⅰ protein. The results showed that a single PCR amplified product was obtained, and the sequence was homologous to the known sequence.. IGF Ⅰ immunostain was positive in the cytoplasm of trabecular meshwork cells. It was concluded that trabecular meshwork cells produce IGF Ⅰ and contribute to the presence of IGF Ⅰ in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by IGF Ⅰ not only through paracrine, but also autocrine action. Whether abnormal down regulations in IGF Ⅰ production may contribute to the pathogenesis of primary open angle glaucoma and the possibility of promoting the autocrine action of IGF Ⅰ by trabecular meshwork cells to treat the diesease is worth further investigation.
文摘Whether transforming growth factor β 2 (TGF β 2) induces apoptosis of human trabecular meshwork cells was investigated in vitro . Cultured 3 5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF β 2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44) %, (4.43 ±1.17) % and (9.60±2.05) % respectively with different concentrations [1 ng/ml ( P< 0.05), 3.2 ng/ml ( P< 0.01)] of TGF β 2 with the difference being significant between experimental group and control group[(1.41±0.34) %]. It was concluded that TGF β 2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.
文摘To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTG-ASDON2 were significantly decreased as compared with that of the controls (P<0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.
文摘The effects of different doses of nitric oxide (NO) on the proliferation and apoptosis of the cultured bovine trabecular meshwork (TM) cells were studied. L arginine and N G nitro L arginine methyl (L NAME) were incubated with TM cells for 48 h. In the control group, no medicine was given. In the experimental groups, concentrations of L arginine and L NAME were 1×10 -7 mol/L, 1×10 -6 mol/L, 1×10 -5 mol/L, 1×10 -4 mol/L, 1×10 -3 mol/L and 1×10 -2 mol/L, respectively. NO 2 - in supernate, the proliferation and apoptosis of TM cells and mRNA expression of bcl 2 and bax were measured by Griess reagent, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), MTT assay and in situ hybridization,respectively. The results showed that L arginine with concentration ≥1×10 -4 mol/L could induce apoptosis of the TM cells and inhibit the proliferation of TM cells through increasing the NO levels, down regulating bcl 2 mRNA expression and up regulating bax mRNA expression; L NAME with concentration ≥1×10 -5 mol/L could induce the proliferation of the TM cells through suppressing the production of NO. It was concluded that NO in high level could induce apoptosis of the TM cells and suppress the proliferation of the TM cells.
